This invention relates to reagents, methods and apparatus for the isolation of cellular components such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins and other materials from natural cellular sources or other sources containing these materials.
Cells contain a wide variety of cellular components appropriate to their function. They contain, for example, DNA and their expression products including a host of proteinaceous materials. This invention is useful for the isolation of such cellular components, but in particular, the invention is principally suited for the isolation of nucleic acids, DNA and RNA.
DNA is a critical component in the sequence of biological reactions which results in the expression of the myriads of proteins including hormones, enzymes and structural tissue essential for the existence of all forms of life. There is a critical need for small and large amounts of DNA for research purposes as well as diagnostic and therapeutic uses.
Plant/animal cells, tissues and organs, insects and microorganisms including viruses, yeast, fungi, algae and bacteria, and other materials are potential sources of DNA. However, the structural organization of some of these sources can be so strong such that it is difficult, time consuming and may require expensive equipment to isolate DNA from those tissues.
For instance, DNA isolation from certain bacteria is difficult because the cell walls are not readily susceptible to lysis. Current protocols for isolating DNA from bacteria frequently employ enzymes such as lysostaphin or lysozyme to digest the bacterial cell wall followed by the addition of denaturing agents to lyse cells and inactivate the nucleases.
The isolation of nucleic acids from various sources, particularly plants, yeast, bacteria, and certain tissues, such as muscle, bone, cartilage, seeds, bark and the like, is difficult due to the presence of cellular structures which protect the tissue, such as rigid cell walls, or other rigid structures, and therefore difficult to rupture completely with commonly used buffers. Removal of these obstacles is tedious and not always feasible with available methods. Variations in nucleic acid yield and quality from the various extraction procedures probably arises from the non-homogeneity (inconsistency) of the tissue as it is broken up. Thus, there is a need for a new technique for disrupting the tissue by a thorough, yet delimited mechanism to allow the rapid isolation of nucleic acids in a reproducible manner without the need to excessively homogenize the cells or tissues.
Procedures have now been discovered which makes possible the separation and isolation of large molecular weight DNA of exceptionally high quality in high yields from a variety of tissues. These procedure are very convenient and can be completed in a very short period of time, typically less than one half hour. This process is, moreover, applicable not only to intact biological tissue but also to microorganisms such as bacteria and yeast, and also to plant tissues as sources of DNA. Such sources, especially bacteria, yeast and plants are much more convenient than complex biological tissue from higher organisms as a source of DNA because they are uniform, readily available in any desired quantities and easier to work with than biological tissue.
The novel procedure of this invention comprises the application of sufficient mechanical energy to the cell walls of the selected DNA source to disrupt the cell walls and release the DNA. The essence of this invention is the discovery of the present methods for tissue or cell disruption in which the tissues and/or cell walls are fractured by specified forces created by the reciprocal motion producing the mechanical energy in a container with the tissue and liquid medium, thereby releasing the DNA from the tissue and into the medium.
In some preferred embodiments, the method includes the use of tissue and/or cell wall fracturing particles in the disruptive media in a closed container.
After lysis of the tissue, the released DNA can be recovered in high yield and purity by any of a variety of recovery methods. Exemplary DNA recovery methods are described further herein.
There are a number of advantages provided by the process of this invention especially when conducted for the isolation of DNA. These include:
1. Applicability to DNA sources such as bacterial cells, fungi, plant cells and other intractable sources which have heretofore been refractory to homogenization procedures with any other extractant media or manipulation.
2. Recovery of DNA as a high yield product substantially uncontaminated by other cellular components.
3. Applicability to the production of both small and large quantities of DNA in batch, multiple sample or continuous processes.
4. Completion in a very short period of time.
5. No ultracentrifugation is required.
6. Isolation of high molecular weight DNA.
7. The reagents used in the methods of the invention are substantially non-toxic, odor free and readily available at commercially attractive prices.